Chromatin insulation orchestrates matrix metalloproteinase gene cluster expression reprogramming in aggressive breast cancer tumors

Abstract

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome contributes to matrix metalloprotease (MMP) dysregulation impacting tumor invasion, which is the first step of the metastatic process.

Methods: We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with MMP gene expression and invasion. We employed CRISPR/Cas9 to disrupt the CCCTC-Binding Factor (CTCF) binding site on an insulator element downstream of the MMP8 gene (IE8) in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. The potential of our findings to predict the progression of ductal carcinoma in situ (DCIS), was tested in data from clinical specimens.

Results: We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was associated with worse relapse-free (HR = 1.57 [1.06 - 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 - 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability and collagen degradation. We observed that the MMP expression pattern predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p < 0.01).

Conclusion: Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer.

Keywords: ATAC-seq; Breast cancer; CTCF; Chromatin; Gene regulatory element; Hi-C; Insulator; Invasion; MMP1; MMP8; RNA-seq; cis-regulatory element.

Fig. 1

MMP expression and its clinical relevance in TNBC. a. Gene expression of MMP genes located on MMP region (chr11q22.2) comparing normal (N = 99) and TNBC (N = 90) patient-derived samples from TCGA consortium. Mann-Whitney test. ns: no significant, *P < 0.05, **P < 0.01. b. Correlation matrix for the expression genes located at the MMP locus located genes (P < 0.01). c. Gene distribution at MMP region (top). Genes were classified according to their location as 5’ or 3’. Gene location was used to define MMP signatures to perform Kaplan-Meier curves of relapse-free survival (left) and overall survival (right)

Fig. 2

Identification and edition of gene regulatory elements at chr11q22.2. a. Scheme of identified enhancer and insulator elements. CRISPR/Cas9-disrupted Insulator element 8 (IE8) is highlighted. b. Expression profile of 5’ and 3‘MMP genes in TNBC cell lines. c. Hi-C contact frequency matrix for the 3 Mb genomic region surrounding IE8 binned at 10-kb resolution. d. CTCF binding is represented as fold enrichment (top) relative to isotype control in MDA-MB-231 and MDA-MB-436 and (bottom) relative fold enrichment after IE8 disruption. Student’s T-test. *P < 0.05, **P < 0.01

Fig. 3

Local consequences of IE8 disruption. a. (top) Description of differentially accessible regions in MDA-MB-231 and MDA-MB-436 upon IE8 disruption and (bottom) representation of commonly modulated regions in both cell lines. b. (top) ATAC-seq peak intensity signals of all the TSS present in the whole genome, chr11 and 10 MB around IE8 (middle) Heatmap of active TSS in TNBC models and (bottom) variations on the accessibility of promoter regions of 5’ MMPs (purple) and 3’MMPs (green) regions in MDA-MB-231 and MDA-MB-436 before and after IE8 disruption. c. Illustrative examples of variation in accessibility upon IE8 disruption (IE8dis minus WT) in the IE8 region (grey), the 5’ MMP region (purple), and the 3’MMP region(green)

Fig. 4

mRNA expression changes after IE8 disruption. a. Volcano plot summarizes the RNA-seq results in MDA-MB-231. 166 genes were significantly upregulated (Padj < 0.05) (blue dots), whereas only 71 were downregulated (red dots). b. Representation factor of differentially expressed genes in MDA-MB-231. Hypergeometric test. ns P > 0.05. c. Variation of MMP8 and MMP1 RNA expression in MDA-MB-231 in RNA-seq. Student’s T-test. *P < 0.05, **P < 0.01. d. MMP1 (top) and MMP8 (bottom) mRNA expression levels determined by qPCR in MDA-MB-231 and MDA-MB-436 models. Student’s T-test. ns P > 0.05, *P < 0.05, **P < 0.01 e. Variation of accessibility in enhancers close to MMP2 (top) and MMP9 (bottom) promoters. f. MMP2 (top) and MMP9 (bottom) mRNA expression levels determined by qPCR in MDA-MB-231 and MDA-MB-436 models. Student’s T-test. ns P > 0.05, *P < 0.05, **P < 0.01

Fig. 5

Functional impact of changes in MMP protein expression and activity after IE8 disruption. Evaluation of MMP8 (a) and MMP1 (b) levels released to the extracellular space after IE8 disruption in MDA-MB-231 and MDA-MB-436. c. Time-course of MMP-1 activity in MDA-MB-231 and MDA-MB-436 upon IE8 disruption. d. (left) An illustrative example of anchorage-independent spheroid growth of MDA-MB-231 and MDA-MB-436 cell models and (right) volume quantification. e. Colorimetric quantification of cell invasion MDA-MB-231 and MDA-MB-436 cell invasion. Student’s T-test. ns: no significant, *P < 0.05, **P < 0.01, ***P < 0.001. O.D. Optical density

Fig. 6

Implications of MMPs in DCIS progression. (a) 3’MMPs/5’MMPs and (b) MMP1/MMP8 ratios of standardized gene expression DCIS and invasive ductal carcinoma. Mann Whitney test. ns: no significant, **P < 0.01. Kaplan-Meier curves of relapse-free survival of DCIS using (c) 3’MMPs/5’MMPs and (d) MMP1/MMP8 ratios. ROC curves displaying the performance of (e) 3’MMPs/5’MMPs and (f) MMP1/MMP8 ratios in in situ ductal carcinoma